This revised R21 application is responsive to PA-11-261 Exploratory/Developmental Research Grant Program and is focused on developing a potential protein biomarker to identify a subset of patients with irritable bowel syndrome (IBS). IBS is common functional bowel disorder characterized by chronic, intermittent abdominal pain and alterations in bowel pattern. Severity of symptoms in IBS range from mild to severe and debilitating. Costs, exceeding $6 billion/year in the US are attributable to the number of diagnostic procedures, clinician visits, medications, and work or school-related absences. IBS affects millions of people in the US with a greater proportion being women. Specifically this application will examine the relationship of urine and stool trefoil factor 3 (TFF3) and symptoms (abdominal pain, stooling pattern) as well as the association of this protein with immune measures and characteristics of stool micro biome. TFF-3 is a protein produced by the goblet cells of the intestinal and colonic mucosa. There is evidence to support its role in the adherence of microbes to the mucus epithelia of the GI. Thus, high levels of urine and stool TTF3 may reflect increased production in response to dysbiosis (imbalanced intestinal flora) and/or alterations in GI barrier function (increased permeability). Using previously collected stool and urine specimens from a prior NINR funded study with carefully phenotyped women with IBS (n=80) we will explore the relationship of TFF3 to stool micro biome composition as determined by bacterial DNA analysis using 454 pyro sequencing pipeline in collaboration with colleagues at Baylor College of Medicine. Multiple analyses will be performed on the 16sRNA gene sequencing data including determination of bacterial diversity, evenness, richness and relative abundance of bacteria identified in each sample. In addition, we will explore the correlation of stool and urine TFF3 levels with permeability as previously measured in these subjects. This study is innovative and high risk in the examination of a protein involved in gut inflammation/protection/repair in concert with the gut micro biome and symptoms.